Title:
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The regulation of the intestinal cancer stem cell marker LGR5 by the dietary fibre derived chemopreventive agent sodium butyrate via an epigenetic mechanism
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The cancer stem cell hypothesis suggests that tumour growth is initiated and maintained
by a subset of tumour cells that display stem cell- like properties. Stem cell markers such
as Leucine-rich repeat-containing G-protein coupled receptor 5 (LGRS), a regulator and
downstream target of Wnt signalling, have been identified in the normal intestinal stem
cells and also in colorectal tumours. This project aimed to investigate the regulation of
LGRS by epigenetic mechanisms and in particular by sodium butyrate, a dietary fibre
derivative, histone deacetylase (HDAC) inhibitor and candidate chemopreventive agent. It
is important to study dietary compounds as many have been reported to play a role in
carcinogenesis. This investigation may therefore lead to improved chemoprevention and
the development of new cancer therapies. Initially the role of DNA methylation in
regulating LGRS expression in normal t issue and cancer cell lines was investigated,
however, although LGRS methylation was observed in some cell lines, no evidence for the
functional regulation of LGRS by methylation was found. This study hypothesised that
butyrate acts as a chemopreventive agent by targeting cancer stem cells and therefore
aimed to investigate the effect of butyrate on the expression of LGRS and other cancer
stem cell markers. Butyrate at physiological concentrations was found to downregulate
LGRS expression in colorectal cancer cell lines through inhibition of LGR5 gene
transcription. This effect occurred within four hours of treatment and was reversible upon
removal of butyrate. LGRS expression was also decreased by other pan and class I HDAC
inhibitors such as Trichostatin A and Mocetinostat, leading to the conclusion that HDAC
inhibit ion was the critical mechanism by which butyrate regulated LGRS. siRNA knockdown
of individual HDACs showed that it was the inhibition of HDACl specifically that mediated
this effect. Additional work in to the mechanism of butyrate action suggested that Wnt
signalling is not involved in the downregulation of LGRS and further work in to other
potential intermediate factors will be interesting. Finally the role of LGRS overexpression
on cell growth and response to butyrate was investigated. These results suggested that in
some cell lines and culture conditions LGRS may enhance cell growth and also reduce the
growth inhibitory effects of butyrate. These results present a novel mechanism for the
regulation of LGRS, an important cancer stem cell marker, by a well-known dietary derived
chemopreventive agent and hint at a role for this effect in regulating stem cell dynamics
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